Preparing Plasmid DNA from Bacteria.

ABSTRACT

The most common method for isolating plasmid DNA is derived from an alkaline lysis procedure. The procedure exploits the differential partitioning of plasmid and chromosomal DNA when denatured by alkali and subsequently renatured by neutralization of the medium. The circular covalently closed nature of plasmid DNA allows the denatured DNA strands to quickly find each other and reanneal during the renaturation step. This is not the case for chromosomal DNA, which, upon neutralization, aggregates with denatured proteins through hydrophobic interactions. As a result, plasmid DNA remains in solution and can be easily separated from most of the other macromolecules that coprecipitate. For the subsequent purification step, one can use the silica membrane technology integrated in many commercial kits. This technology exploits the ability of DNA to bind to silica in the presence of chaotropic salts. DNA is retained by a silica-based column, whereas most of the polysaccharides and proteins flow through. After wash steps to eliminate residual contaminants and salts, DNA is selectively eluted under low-salt conditions. A kit-free but relatively more cumbersome alternative to this procedure is the traditional phenol-chloroform extraction method followed by ethanol precipitation. Both methods are detailed here.